Tumor necrosis factor α (TNF-α or TNF-alpha) is a pleiotropic cytokine that is primarily produced by activated macrophages and lymphocytes; but is also expressed in endothelial cells and other cell types. TNF-alpha is a major mediator of inflammatory, immunological, and pathophysiological reactions. (Grell, M., et al., (1995) Cell, 83:793-802). Two distinct forms of TNF exist, a 26 kDa membrane expressed form and the soluble 17 kDa cytokine which is derived from proteolytic cleavage of the 26 kDa form. The soluble TNF polypeptide is 157 amino acids long and is the primary biologically active molecule.
TNF-alpha exerts its biological effects through interaction with high-affinity cell surface receptors. Two distinct membrane TNF-alpha receptors have been cloned and characterized. These are a 55 kDa species, designated p55 TNF-R and a 75 kDa species designated p75 TNF-R (Corcoran. A. E., et al., (1994) Eur. J. Biochem., 223:831-840). The two TNF receptors exhibit 28% similarity at the amino acid level. This is confined to the extracellular domain and consists of four repeating cysteine-rich motifs, each of approximately 40 amino acids. Each motif contains four to six cysteines in conserved positions. Dayhoff analysis shows the greatest intersubunit similarity among the first three repeats in each receptor. This characteristic structure is shared with a number of other receptors and cell surface molecules, which comprise the TNF-R/nerve growth factor receptor superfamily (Corcoran. A. E., et al., (1994) Eur. J. Biochem., 223:831-840).
TNF signaling is initiated by receptor clustering, either by the trivalent ligand TNF or by cross-linking monoclonal antibodies (Vandevoorde, V., et al., (1997) J. Cell Biol., 137:1627-1638).
Crystallographic studies of TNF and the structurally related cytokine, lymphotoxin (LT) have shown that both cytokines exist as homotrimers, with subunits packed edge to edge in a threefold symmetry. Structurally, neither TNF or LT reflect the repeating pattern of the their receptors. Each monomer is cone shaped and contains two hydrophilic loops on opposite sides of the base of the cone. Recent crystal structure determination of a p55 soluble TNF-R/LT complex has confirmed the hypothesis that loops from adjacent monomers join together to form a groove between monomers and that TNF-R binds in these grooves (Corcoran. A. E., et al., (1994) Eur. J. Biochem., 223:831-840).
Random mutagenesis has been used to identify active sites in TNF-alpha responsible for the loss of cytotoxic activity (Van Ostade, X., et al., (1991) EMBO J., 10:827-836). Human TNF muteins having higher binding affinity for human p75-TNF receptor than for human p55-TNF receptor have also been disclosed (U.S. Pat. No. 5,597,899 and Loetscher et al., J. Biol. Chem., 268(35) pp263050-26357 (1993)).
The key role played by TNF-alpha in inflammation, cellular immune responses and the pathology of many diseases has led to the search for antagonists of TNF-alpha. Soluble TNF receptors which interfere with TNF-alpha signaling have been isolated and are marketed by Immunex as Enbrel® (entanercept) for the treatment of rheumatoid arthritis. The side effect profiles of entarecept, Remicade® (infliximab) and Humira® (adalimumab) all show an increased likelihood of infection (e.g., listeriosis) and tuberculosis and other infections. (See, Slifman, N R, et al, “Listeria monocytogene Infection as a Complication of Treatment with Tumor Necrosis Factor α-Neutralizing Agents”, Arthritis & Rheumatism, 48(2): 319-324 (2003) and Olleros, M L, et al., “Transmembrane TNF Induces an Efficient Cell-Mediated Immunity and Resistance to Mycobacterium bovis Bacillus Calmette-Guerin Infection in the Absence of Secreted TNF and Lymphotoxin-α, J. Immunol., 168: 3394-3401 (2002); and Granulomatous Infectious Diseases Associated with Tumor Necrosis Factor Antagonists, R. S. Wallis, et al., Clinical Infectious Diseases 2004; 38:1261-5).
In view of the serious side effects of existing therapies, a therapeutic that is more potent and has a reduced side effect profile is still needed.